DNA replication, repair and recombination 2 (lecture 4) Flashcards by Tyler Turner

need to repair damaged DNA, not only from replication errors but ___ that occur in the genome

accidental lesions

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Fewer than _____ accidental base changes result in a permanent mutation

1/1000

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5000 purine bases are lost every day due to a spontaneous reaction called ______

depurination

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Spontaneous ______ of C to U occurs at 100 bases/day

Deamination

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Depurination is a result of hydrolysis of the _____ linkage

N-glycosyl linkage (this is the bond that attaches the base to the sugar)

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DNA damage can occur from exposure to reactive forms of ___ in the cell or chemicals in environment

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UV radiation from sun can produce a covalent linkage between two adjacent pyrimidines (T-T or C-T) this is known as

pyrimidine dimers

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if unprepared when DNA is replicated DNA damage can lead to with a ___ or a _____ in the daughter strand

deletion or base pair substitution

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what are the 4 types of DNA repair

Base excision repair
Nucleotide excision repair
Transcription-coupled repair
Double-Strand break repair

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What ar the two types of double-stranded break repairs

Non-homologous end joining

- homologous recombination

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There are atleast ____ different types of DNA glycosylases

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each of the 6 different types of DNA glycosylases recognizes a specific type of ____ and catalyzes it ____

altered base, removal

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what type of repair is enzyme mediated “flipping out” of base from the helix

Base excision repair

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Explain Base excision repair

-DNA glycosylases travel along DNA using base-flipping to evaluate the status of each base. once the enzyme finds the damaged base that it recognizes, it removes that base from its sugar. The “missing tooth” created by DNA glycosylase and the enzymes AP endonuclease and phosphodiesterase cut the phosphodiester backbone. DNA polymerase adds new nucleotides and DNA ligase seals the nick

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once DNA glycosylase finds an incorrect base it cleaves the ___ bond connecting the base with sugar

glycosyl

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______ are directly repaired beginning with Ap endonuclease

Depurinations

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How does nucleotide excision repair differ from base excision repair

in how damage is removed

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Nucleotide excision repair can repair any ____ lesion like those chemically-induced and various pyrimidine dimers

bulky

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explain Nucleotide excision pathway

a large multi enzyme complex scans the DNA for a distortion in the double helix, rather than for a specific base change. once it finds a lesion, Excision nuclease cleaves the phosphodiester backbone of the abnormal strand on both sides of the distortion, and a DNA helicase peels away the single-stranded oligonucleotide containing the lesion. the large gap produced in the DNA helix is then repaired by DNA polymerase and DNA ligase

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What did Tomas Lindahl win the 2015 nobel prize in chemistry for

he identified the proteins involved in the base excision repair system
he identified the first glycosylase

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What did Aziz Sancar win the 2015 nobel prize in chemistry for

identified the proteins involved in nucleotide excision repair system
identified an enzyme he called photolyase

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Cells can preferentially direct DNA repair to sequences that are being actively transcribed by linking ____ with DNA repair

RNA polymerase

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The importance of of transcription-coupled excision repair is seen in people with ______ syndrome, which is caused by a defect in this coupling. These individuals suffer from growth retardation, skeletal abnormalities, progressive neural retardation, and severe sensitivity to sunlight. RNA polymerase molecules are permanently stalled at sites of DNA damage

Cockayne

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RNA polymerase stalls at lesions and directs

repair machinery there

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_______ works with BER, NER and others to repair genes that are being expressed when the damage occurs

Transcription-coupled repair

Transcription-coupled repair is specific for the

strand being transcribed (the non-transcribed strand is repaired at the same rate as DNA not being transcribed)

Cockayne's syndrome

- Defect in transcription-coupled repair - Growth retardation, Skeletal abnormalities, and sensitivity to sunlight - RNA polymerase is permanently stalled at sites of damage in important genes

______ is the simplest purine base capable of pairing specifically with Cytosine

hypoxanthine

Hypoxanthine is the direct deamination product of

adenosine

is the DNA molecule optimally constructed for repair? explain

- Yes - 2 strands means there is a backup copy - Nature of 4 buses makes distinction between damaged/undamaged obvious - Every deamination event forms unnatural base - Possible reason why RNA is not the hereditary information. (cannot distinguish between deaminated Cytosine and natural uracil)

Deamination of guanine yields

Xanthine

Deamination of cytosine yields

Uracil

Deamination of thymine yields

there is no deamination of thymine because it does not have the amine side group

What is a possible reason why RNA is not the hereditary information

- RNA cannot distinguish between deaminated Cytosine and natural Uracil

Special problem exists with methylated ______ in vertebrate DNA

cytosines

methylated cytosines occurs at some ____ sequences and associated with ______

CpG sequences, inactive genes

Deamination of methyl-Cytosine produces ___ mismatch with ____

natural T mismatched with G

about 3% of the C nucleotides in vertebrate DNAs are methylated to help in

controlling gene expression

only about 3% of cytosine nucleotides in the human genome are methylated but they account for ____ of all point mutations associated with inherited human diseases

1/3

A special DNA glycosylase recognizes and removes the Thymine that is made when methylated cytosines are deaminated (note that this thymine is thus mismatched with G) but the repair is relatively

ineffective

_____ are used when the cell has sustained extensive damage

Translesion Polymerases

less accurate, backup polymerases to repair damage

Translesion polymerases

The highly accurate replicative DNA polymerases stall when they encounter damaged DNA, and in emergencies cells employ versatile, but less accurate, backup polymerases, known as _______, to replicate through the DNA damage

translesion polymerases

humans have ____ different types of translesion polymerases

some types of translesion polymerases recognize a specific type of DNA damage and correctly add the nucleotide recede to restore the initial sequence. Other make only ______, especially when the template base has been extensively damaged

good-guesses

Translesion polymerases are not as accurate as the normal replicative polymerases when they copy a normal DNA sequence. because they lack ______ and they ____

they lack exonucleolytic proofreading activity; in addition many are much less discriminating than the replicative polymerase in choosing which nucleotide to incorporate initially.

Because of Translesion polymerases inaccuracy they only add a couple of nucleotides before

the replicative polymerase reassociates

Explain Translesion polymerase use in repairing DNA

A replicative polymerase stalled at a site of DNA damage is recognized by the cell as needing rescue. Specialized enzymes covalently modify the sliding clamp (typically, it is ubiquitylated) which releases the replicative DNA polymerase and, together with damaged DNA, attracts a translesion polymerase specific to that type of damage. once the damaged DNA is bypassed, the covalent modification of the clamp is removed, the translesion polymerase dissociates, and the replicative polymerase is brought back into play

Causes of double-strand breaks

- Ionizing radiation - replication errors - oxidizing agents and other metabolites

if double-stranded breaks are left unrepaired chromosomes will

break into smaller fragments and be lost

What are two types of double strand break repair

- non-homologous end joining | - Homologous recombination

_______ brings broken ends together and rejoins by DNA ligation; one or more nucleotides will be lost

non-homologous end joining

what type of double strand break repair predominates in human somatic cells and is generally okay since so little of genome is protein coding

non-homologous end joining

By the time a human reaches the age of 70, the typical somatic cell contains over _____ such scars, distributed throughout its genome, representing places where DNA has been inaccurately repaired by non homologous end joining

2000

explain non homologous end joining

used to fix double strand breaks (usually takes place when cells have not yet duplicated their DNA) Double stranded break leads to nuclease processing both the DNA ends (this is because often the nucleotides around the break are damaged or altered) the sites are then rejoined by DNA ligation

_____ ensure the completion of one stage in the cell cycle before the next can begin

Checkpoints

Presence of DNA damage triggers various checkpoints such as

- blocks entry from G1 into S phase - Slows down progression through S phase - blocks transition form G2 to M phase

Checkpoints give the cell extra time to

repair DNA damage

a kinase that generates intracellular signals that alert the cell to DNA damage and up regulate expression of DNA repair genes

ATM protein

Mutation in ATM protein lead to

ataxia telengiectasia (AT)

Ataxia telengiectasia (AT)

- caused by mutations in ATM protein | - symptoms include neurodegeneratiion, predisposition to cancer, genome instability due to unrepaired DNA lesions

Genetic exchange between a pair of homologous DNA sequences

Homologous recombination

functions of homologous recombination

- repair of double-standed breaks - especially at stalled or broken replication forks - Exchange of genetic information to create new combinations of genetic sequences - crossing over and gene conversion in meiosis - Mechanical role in assuring accurate chromosome segregation

Homologous recombination is a fundamental process and common to

all organisms

_____ guides homologous recombination

base-pairing

DNA duplexes ___ each other looking for regions of homology

"sample"

DNA double helix reforming from its separated single strands

Hybridization (also called renaturation)

Once a region of homology is found

the single strands rapidly pair up

base-pairing of homologous recombination can create a double helix from strands that originate from different molecules but are complementary to one another (or atleast largely complementary) is called

heteroduplex DNA

Explain repairing double stranded breaks by homologous recombination

- 5' ends degraded by exonuclease - one 3' end invades homologous template and primes repair DNA synthesis - The newly synthesized 3' end of the invading strand is then able to anneal to the other original 3' overhang in the damaged chromosome through complementary base pairing - gaps are filled in and ligated

DNA hybridization requires a _____

single-stranded DNA (freed from pairing with complement so it can pair with the 2nd strand)

DNA strand exchange is carried out by the _____ protein

RecA/Rad51

The single-stranded invading strand is directed by _______ and other proteins

RecA (bacteria) Rad51 in eukaryotes

RecA/Rad51 binds cooperatively to single stranded DNA and holds it in a unique configuration, with the backbone _____

stretched out

RecA/Rad51 moves the single strand down the duplex searching ____ nucleotides at a time

Once RecA/Rad51 has identified a homologous sequence strand invasion occurs, forming a heteroduplex. This invasion requires an extended stretch of how many homologous base pairs

at least 15

_____ can rescue broken DNA replication forks

Homologous Recombination

The first step in repairing a broken replication fork with homologous recombination is after the replication fork breaks the

exonuclease degrades 5' end

what is the second step in homologous recombination repair of a broken replication fork (caused by a nick in one strand)

Strand invasion (note this is after exonuclease degrade the 5' end of the broken strand). This pairs single-stranded DNA with complementary strand in different double-stranded helix. Forms a region of heteroduplex DNA

Accurate repair process can still cause problems for the cell . such as

- use of a non-functioning homolog to "repair" the other homolog - loss of heterozygosity (this is a critical first step in cancer development and rare occurrence)

Processing of broken ends is coordinated with the ____

cell cycle

Nucleases for generating 3' invading strand are only active in _______ and ____ phase

S and G2 phase

Nucleases for generating 3' invading strand are only active in S and G2 phase this ensures a ______ or ____ will be the most likely template for repair

replicated chromosome or sister chromatid

Give examples of how the cell is able to prevent DNA repair in the absence of damage

- Loading of RecA on DNA is tightly controlled | - Repair proteins dispersed throughout cell

mutations in proteins involved in recombination can cause

cancer

mutations in ____ and ____ lead to increased rates of breast cancer

Brca1 and Brca2

_____ regulates the processing of broken ends of chromosome

Brca1

____ maintains Rad51 (RecA) inactive until it is at site of damage

Brca2

Brca1 regulates the processing of broken ends of chromosomes. A mutation in this protein leads to

use of non-homologous end-joining processes

Brac2 maintains Rad51 (RecA) inactive until it is safe at site of damage. mutations in this can lead to

- Rad51 (RecA) an become active at times when not desired

what type of double-strand break repair process does not require a template, creates mutation at site of repair, and can also create translocations

non-homologous end joining

What type of double-strand break repair process use daughter strand DNA duplex as template, no loss or alteration of DNA at repair site, Can repair other types of DNA damage, mechanism and proteins conserved in all organisms

Homologous recombination

____ complex recognizes double-stranded breaks and mobilizes additional proteins to repair the damage

Mre11 (this is a nuclease that processes damaged DNA in preparation for homologous recombination)